The extracellular calcium ion regulates a late stage of prereplicative development of non-neoplastic cells through a calcium, Ca-calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase mechanism. This regulatory system is somehow altered in neoplastic cells because they need 50-100-fold less extracellular calcium to proliferate than non-neoplastic cells. Of the 25 cell lines tested, 15 formed tumors in arrhythmic nude mice, and formed colonies in soft agar and low (0.02 mM) calcium medium. Ten cell lines did not form tumors in arrhythmic nude mice nor form colonies in soft agar a low-calcium medium. Cells treated with a chemical carcinogen (MNNG) and subsequent transformation can be selected by incubating the cultures in a calcium-deficient medium. This assay is being further developed. The blockage of non-neoplastic cell proliferation in low-calcium medium can be overcome by calcium or a variety of other agents which all seem to function by causing a brief cyclic AMP surge. Tumor-promoting agents (such as TPA, saccharin, cyclamates, phenobarbital, PGE, and EGF) can also replace calcium and just as rapidly (within one hr.) stimulate DNA synthesis. The mechanism of this increased DNA-synthetic activity is being investigated.